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Integrin/Fak/Src-mediated regulation of cell survival and anoikis in human intestinal epithelial crypt cells: selective engagement and roles of PI3-K isoform complexes.

Beauséjour M, Noël D, Thibodeau S, Bouchard V, Harnois C, Beaulieu JF, Demers MJ, Vachon PH - Apoptosis (2012)

Bottom Line: Class I PI3-K consists of a complex formed by a catalytic (C) and regulatory (R) subunit.Three R (p85α, β, and p55γ) and four C (p110α, β, γ and δ) isoforms are known.We report that: (1) the predominant PI3-K complexes expressed by HIEC cells are p110α/p85β and p110α/p55γ; (2) the inhibition and/or siRNA-mediated expression silencing of p110α, but not that of p110β, γ or δ, results in Akt-1 down-activation and consequent apoptosis; (3) the expression silencing of p85β or p55γ, but not that of p85α, likewise induces Akt-1 down-activation and apoptosis; however, the impact of a loss of p55γ on both Akt-1 activation and cell survival is significantly greater than that from the loss of p85β; and (4) both the p110α/p85β and p110α/p55γ complexes are engaged by β1 integrin/Fak/Src signaling; however, the engagement of p110α/p85β is primarily Src-dependent, whereas that of p110α/p55γ is primarily Fak-dependent (but Src-independent).

View Article: PubMed Central - PubMed

Affiliation: Département d'anatomie et de Biologie Cellulaire, Faculté de Médecine et des Sciences de la Santé, Université de Sherbrooke, Sherbrooke, QC, Canada.

ABSTRACT
In human intestinal epithelial crypt (HIEC) cells, the PI3-K/Akt-1 pathway is crucial for the promotion of cell survival and suppression of anoikis. Class I PI3-K consists of a complex formed by a catalytic (C) and regulatory (R) subunit. Three R (p85α, β, and p55γ) and four C (p110α, β, γ and δ) isoforms are known. Herein, we analyzed the expression of PI3-K isoforms in HIEC cells and determined their roles in cell survival, as well as in the β1 integrin/Fak/Src-mediated suppression of anoikis. We report that: (1) the predominant PI3-K complexes expressed by HIEC cells are p110α/p85β and p110α/p55γ; (2) the inhibition and/or siRNA-mediated expression silencing of p110α, but not that of p110β, γ or δ, results in Akt-1 down-activation and consequent apoptosis; (3) the expression silencing of p85β or p55γ, but not that of p85α, likewise induces Akt-1 down-activation and apoptosis; however, the impact of a loss of p55γ on both Akt-1 activation and cell survival is significantly greater than that from the loss of p85β; and (4) both the p110α/p85β and p110α/p55γ complexes are engaged by β1 integrin/Fak/Src signaling; however, the engagement of p110α/p85β is primarily Src-dependent, whereas that of p110α/p55γ is primarily Fak-dependent (but Src-independent). Hence, HIEC cells selectively express PI3-K isoform complexes, translating into distinct roles in Akt-1 activation and cell survival, as well as in a selective engagement by Fak and/or Src within the context of β1 integrin/Fak/Src-mediated suppression of anoikis.

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β1 integrin/Fak/Src-mediated signaling requirement for anoikis suppression in HIEC cells. a Representative (n ≥ 5) CAD-mediated DNA laddering assays from HIEC cell control cultures or cultures treated with PF573228, PP2, non-immune IgGs, the β1 integrin-blocking antibody P4C10, or kept in suspension in polyHEMA-coated dishes (suspension). L 100-bp DNA size markers. b HIEC cell cultures were maintained as in (a), except without the suspension treatment. ISEL was then performed. c HIEC cell cultures were maintained as in (a), except without the IgGs and P4C10 treatments. CASP-3 relative activity was then established, using the substrate Ac-DEVD-AMC, by comparison to controls. b, c Statistically significant (0.0001 ≤ P ≤ 0.001) differences between treated and control cultures are indicated by (*)
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Fig5: β1 integrin/Fak/Src-mediated signaling requirement for anoikis suppression in HIEC cells. a Representative (n ≥ 5) CAD-mediated DNA laddering assays from HIEC cell control cultures or cultures treated with PF573228, PP2, non-immune IgGs, the β1 integrin-blocking antibody P4C10, or kept in suspension in polyHEMA-coated dishes (suspension). L 100-bp DNA size markers. b HIEC cell cultures were maintained as in (a), except without the suspension treatment. ISEL was then performed. c HIEC cell cultures were maintained as in (a), except without the IgGs and P4C10 treatments. CASP-3 relative activity was then established, using the substrate Ac-DEVD-AMC, by comparison to controls. b, c Statistically significant (0.0001 ≤ P ≤ 0.001) differences between treated and control cultures are indicated by (*)

Mentions: As expected from our previous studies [28–33, 39], abundant CAD-mediated internucleosomal DNA fragmentation was observed in HIEC-6 cultures that were maintained in suspension (Fig. 5a, suspension), as well as in cultures exposed to the β1 integrin binding activity-blocking P4C10 antibody (Fig. 5a, P4C10), in contrast to control (adhering) cultures (Fig. 5a) and/or those exposed to non-immune IgGs (Fig. 5a, IgGs). Similarly, such DNA laddering was observed when the tyrosine kinase activities of Fak (Fig. 5a, PF573228) or Src (Fig. 5a, PP2) were inhibited. These observations were confirmed by ISEL (Fig. 5b) and CASP-3 activity (Fig. 5c) assays. The relative activation levels of Fak, Src and Akt-1 were then verified in both control and treated cultures. As previously reported [28–33, 39], the inhibition of Fak resulted in a significant down-activation of Fak itself (Fig. 6a, PF573228; 6b, PF573228, open column), of Src (Fig. 6a, PF573228; 6b, PF573228, grey column), and of Akt-1 (Fig. 6a, PF573228; 6b, PF573228, filled column), in a similar fashion as when cells were kept in suspension or exposed to the P4C10 antibody (Fig. 6a, b, suspension, P4C10). In the same vein, the inhibition of Src resulted in its own down-activation (Fig. 6a, PP2; 6b, PP2, grey column) and that of Akt-1 (Fig. 6a, PP2; 6b, PP2, filled column). However, such inhibition of Src had no significant impact on the activation levels of Fak (Fig. 6a, PP2; 6b, PP2, open column). Hence, while these results further support our previous demonstration that the PI3-K/Akt-1 pathway is engaged by integrin β1/Fak/Src signaling in the suppression of anoikis in HIEC cells [28–32, 39], these also suggest that the contributions of Src in such signaling may be primarily Fak-dependent—as observed in other cell contexts [6, 8, 14, 15].Fig. 5


Integrin/Fak/Src-mediated regulation of cell survival and anoikis in human intestinal epithelial crypt cells: selective engagement and roles of PI3-K isoform complexes.

Beauséjour M, Noël D, Thibodeau S, Bouchard V, Harnois C, Beaulieu JF, Demers MJ, Vachon PH - Apoptosis (2012)

β1 integrin/Fak/Src-mediated signaling requirement for anoikis suppression in HIEC cells. a Representative (n ≥ 5) CAD-mediated DNA laddering assays from HIEC cell control cultures or cultures treated with PF573228, PP2, non-immune IgGs, the β1 integrin-blocking antibody P4C10, or kept in suspension in polyHEMA-coated dishes (suspension). L 100-bp DNA size markers. b HIEC cell cultures were maintained as in (a), except without the suspension treatment. ISEL was then performed. c HIEC cell cultures were maintained as in (a), except without the IgGs and P4C10 treatments. CASP-3 relative activity was then established, using the substrate Ac-DEVD-AMC, by comparison to controls. b, c Statistically significant (0.0001 ≤ P ≤ 0.001) differences between treated and control cultures are indicated by (*)
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Fig5: β1 integrin/Fak/Src-mediated signaling requirement for anoikis suppression in HIEC cells. a Representative (n ≥ 5) CAD-mediated DNA laddering assays from HIEC cell control cultures or cultures treated with PF573228, PP2, non-immune IgGs, the β1 integrin-blocking antibody P4C10, or kept in suspension in polyHEMA-coated dishes (suspension). L 100-bp DNA size markers. b HIEC cell cultures were maintained as in (a), except without the suspension treatment. ISEL was then performed. c HIEC cell cultures were maintained as in (a), except without the IgGs and P4C10 treatments. CASP-3 relative activity was then established, using the substrate Ac-DEVD-AMC, by comparison to controls. b, c Statistically significant (0.0001 ≤ P ≤ 0.001) differences between treated and control cultures are indicated by (*)
Mentions: As expected from our previous studies [28–33, 39], abundant CAD-mediated internucleosomal DNA fragmentation was observed in HIEC-6 cultures that were maintained in suspension (Fig. 5a, suspension), as well as in cultures exposed to the β1 integrin binding activity-blocking P4C10 antibody (Fig. 5a, P4C10), in contrast to control (adhering) cultures (Fig. 5a) and/or those exposed to non-immune IgGs (Fig. 5a, IgGs). Similarly, such DNA laddering was observed when the tyrosine kinase activities of Fak (Fig. 5a, PF573228) or Src (Fig. 5a, PP2) were inhibited. These observations were confirmed by ISEL (Fig. 5b) and CASP-3 activity (Fig. 5c) assays. The relative activation levels of Fak, Src and Akt-1 were then verified in both control and treated cultures. As previously reported [28–33, 39], the inhibition of Fak resulted in a significant down-activation of Fak itself (Fig. 6a, PF573228; 6b, PF573228, open column), of Src (Fig. 6a, PF573228; 6b, PF573228, grey column), and of Akt-1 (Fig. 6a, PF573228; 6b, PF573228, filled column), in a similar fashion as when cells were kept in suspension or exposed to the P4C10 antibody (Fig. 6a, b, suspension, P4C10). In the same vein, the inhibition of Src resulted in its own down-activation (Fig. 6a, PP2; 6b, PP2, grey column) and that of Akt-1 (Fig. 6a, PP2; 6b, PP2, filled column). However, such inhibition of Src had no significant impact on the activation levels of Fak (Fig. 6a, PP2; 6b, PP2, open column). Hence, while these results further support our previous demonstration that the PI3-K/Akt-1 pathway is engaged by integrin β1/Fak/Src signaling in the suppression of anoikis in HIEC cells [28–32, 39], these also suggest that the contributions of Src in such signaling may be primarily Fak-dependent—as observed in other cell contexts [6, 8, 14, 15].Fig. 5

Bottom Line: Class I PI3-K consists of a complex formed by a catalytic (C) and regulatory (R) subunit.Three R (p85α, β, and p55γ) and four C (p110α, β, γ and δ) isoforms are known.We report that: (1) the predominant PI3-K complexes expressed by HIEC cells are p110α/p85β and p110α/p55γ; (2) the inhibition and/or siRNA-mediated expression silencing of p110α, but not that of p110β, γ or δ, results in Akt-1 down-activation and consequent apoptosis; (3) the expression silencing of p85β or p55γ, but not that of p85α, likewise induces Akt-1 down-activation and apoptosis; however, the impact of a loss of p55γ on both Akt-1 activation and cell survival is significantly greater than that from the loss of p85β; and (4) both the p110α/p85β and p110α/p55γ complexes are engaged by β1 integrin/Fak/Src signaling; however, the engagement of p110α/p85β is primarily Src-dependent, whereas that of p110α/p55γ is primarily Fak-dependent (but Src-independent).

View Article: PubMed Central - PubMed

Affiliation: Département d'anatomie et de Biologie Cellulaire, Faculté de Médecine et des Sciences de la Santé, Université de Sherbrooke, Sherbrooke, QC, Canada.

ABSTRACT
In human intestinal epithelial crypt (HIEC) cells, the PI3-K/Akt-1 pathway is crucial for the promotion of cell survival and suppression of anoikis. Class I PI3-K consists of a complex formed by a catalytic (C) and regulatory (R) subunit. Three R (p85α, β, and p55γ) and four C (p110α, β, γ and δ) isoforms are known. Herein, we analyzed the expression of PI3-K isoforms in HIEC cells and determined their roles in cell survival, as well as in the β1 integrin/Fak/Src-mediated suppression of anoikis. We report that: (1) the predominant PI3-K complexes expressed by HIEC cells are p110α/p85β and p110α/p55γ; (2) the inhibition and/or siRNA-mediated expression silencing of p110α, but not that of p110β, γ or δ, results in Akt-1 down-activation and consequent apoptosis; (3) the expression silencing of p85β or p55γ, but not that of p85α, likewise induces Akt-1 down-activation and apoptosis; however, the impact of a loss of p55γ on both Akt-1 activation and cell survival is significantly greater than that from the loss of p85β; and (4) both the p110α/p85β and p110α/p55γ complexes are engaged by β1 integrin/Fak/Src signaling; however, the engagement of p110α/p85β is primarily Src-dependent, whereas that of p110α/p55γ is primarily Fak-dependent (but Src-independent). Hence, HIEC cells selectively express PI3-K isoform complexes, translating into distinct roles in Akt-1 activation and cell survival, as well as in a selective engagement by Fak and/or Src within the context of β1 integrin/Fak/Src-mediated suppression of anoikis.

Show MeSH
Related in: MedlinePlus